Intracellular pathogens cause a staggering number of human infections and deaths. The ability to subvert host innate immune responses is a key factor in the establishment of infections by these pathogens. Listeria monocytogenes is a facultative intracellular bacterial pathogen of humans and animals and is frequently used as a model to dissect mechanisms of immunity and pathogenesis. Our prior studies with L. monocytogenes identified a bacterial protein, p60, whose expression and secretion from the bacterium is required for pathogenicity in the mouse model of systemic infection. p60 is not required for Lm infection in cultured cells. We found that p60 stimulates the activation of host natural killer (NK) cells, an immune cell population associated with increased host susceptibility to L. monocytogenes and several other pathogenic bacteria. We have shown that p60 stimulates NK cells by binding to dendritic cells, and eliciting the production of IL-1 and IL-18. The ability of p60 to activate DCs and NK cells maps to a region of the protein containing a LysM domain. Purified p60 protein, or just the region of p60 containing the LysM domain is sufficient to stimulate NK cell activation. The host factor NLRP3 is also required for IL-18 production and NK cell activation in cultures with DCs, NK cells, and p60. In our first Aim, we will further define the mechanisms by which the LysM domain containing peptide uniquely binds and stimulates DCs for IL-18 production and NK cell activation. Molecular, imaging, immunological, and cell biological approaches will be used. In our second Aim, we will investigate how NLRP3 impacts host resistance during systemic bacterial infections. In our third Aim, we will characterize NK cells producing IL-10 in response to L. monocytogenes infection and p60. We also test how IL-10 production by NK cells impacts host immune responses and susceptibility to bacterial infection. Together, our work will reveal how LysM-containing bacterial virulence factors subversively activate specific aspects of innate immunity in order to promote the establishment of systemic infections. PUBLIC HEALTH RELEVANCE: Our studies will investigate the mechanisms by which a secreted bacterial protein activates host innate immune responses, and the basis by which such activation exacerbates infection by the bacterium Listeria monocytogenes. Findings from our work may lead to novel therapies targeted at preventing subversion of host immune responses by this and other pathogens. The work here may also reveal approaches to therapeutically manipulate innate immunity during non-infectious diseases.